Nucleic Acid

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Nucleic Acid
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The Central Dogma
Replication
Transcription
Translation
The flow of genetic information is
unidirectional, from DNA to protein with
messenger RNA as an intermediate.
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DNA Replication

• - the process of making new copies of the DNA
  molecules

Potential mechanisms
organization of DNA strands
Conservative old/old
new/new Semiconservative old/new
new/old Dispersive mixed old and new on each
strand
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Meselson and Stahls replication experiment
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Replication as a process

• 1. Double-stranded DNA unwinds.

2. The junction of the unwound molecules is a
replication fork.
3. A new strand is formed by pairing
complementary bases with the old strand.
4. Two molecules are made. Each has one new and
one old DNA strand.
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Enzymes in DNA replication
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Replication
Helicase protein binds to DNA sequences called
origins and unwinds DNA strands.
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Replication
DNA polymerase enzyme adds DNA nucleotides to
the RNA primer.
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Replication
Leading strand synthesis continues in a 5 to 3
direction.
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Replication
Leading strand synthesis continues in a 5 to 3
direction.
Discontinuous synthesis produces 5 to 3 DNA
segments called Okazaki fragments.
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Replication
Overall direction of replication
3
5
3
5
Okazaki fragment
3
5
5
3
3
5
Leading strand synthesis continues in a 5 to 3
direction.
Discontinuous synthesis produces 5 to 3 DNA
segments called Okazaki fragments.
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Replication
3
5
3
5
3
5
3
5
3
3
5
5
Leading strand synthesis continues in a 5 to 3
direction.
Discontinuous synthesis produces 5 to 3 DNA
segments called Okazaki fragments.
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Replication
3
3
5
Leading strand synthesis continues in a 5 to 3
direction.
Discontinuous synthesis produces 5 to 3 DNA
segments called Okazaki fragments.
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Replication
Exonuclease enzymes remove RNA primers.
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Replication
Exonuclease enzymes remove RNA primers.
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Replication
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Transcription
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Prokaryotic Gene Structure
Promoter CDS Terminator
UTR UTR
Genomic DNA
transcription
mRNA
translation
protein
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Eukaryotic Gene Structure
5 - Promoter Exon1 Intron1
Exon2 Terminator 3
UTR splice splice UTR
transcription
Poly A
translation
protein
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Transcription initiation and elongation
1. Genes need to be expressed to be genes
2. Transcription is directed to specific
locations (promoters)
3. RNA is elongated in the 5-to-3 direction
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Promoter

• Promoter determines
• Which strand will serve as a template.
• Transcription starting point.
• Strength of polymerase binding.
• Frequency of polymerase binding.

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Prokaryotic Promoter

• One type of RNA polymerase.
• Pribnow box located at 10 (6-7bp)
• 35 sequence located at -35 (6bp)

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Eukaryote Promoter

• 3 types of RNA polymerases are employed in
  transcription of genes
• RNA polymerase I transcribes rRNA
• RNA polymerase II transcribes all genes coding
  for polypeptides
• RNA polymerase III transcribes small
  cytoplasmatic RNA, such as tRNA.

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Eukaryote Promoter

• Goldberg-Hogness or TATA located at 30
• Additional regions at 100 and at 200
• Possible distant regions acting as enhancers or
  silencers (even more than 50 kb).

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RNA Synthesis

• DNA template 3-to-5
• RNA synthesis 5-3 no primer needed

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Termination Sites

• The newly synthesized mRNA forms a stem and loop
  structure (lollipop).
• A disassociation signal at the end of the gene
  that stops elongating and releases RNA
  polymerase.
• All terminators (eukaryotes and prokaryotes) form
  a secondary structure.

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Termination Sites

• The terminator region pauses the polymerase and
  causes disassociation.

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Splice Sites

• Eukaryotics only
• Removing internal parts of the newly transcribed
  RNA.
• Takes place in the cell nucleus (hnRNA)

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Splice Sites

• Conserved splice sites are shared by both the
  exon and the intron.
• Different signals on the donor site (3) and on
  the acceptor site (5).

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Translation
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Genetic Terminology
Chromosome - threadlike structures in the
nucleus that carry genetic information Gene -
fundamental unit of heredity - inherited
determinant of a phenotype - sequence
of DNA that instructs a cell to produce a
particular protein DNA -
deoxyribonucleic acid, - the genetic
material - the biochemical that forms genes
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Open Reading Frames (ORF)
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TRANSCRIPTION
Unwinding of gene regions of a DNA molecule
Pre mRNA Transcript Processing
mRNA
rRNA
tRNA
protein subunits
Mature mRNA transcripts
ribosomal subunits
mature tRNA
Convergence of RNAs
TRANSLATION
Cytoplasmic pools of amino acids, tRNAs, and
ribosomal subunits
Synthesis of a polypetide chain at binding sites
for mRNA and tRNA on the surface of an intact
ribosome
FINAL PROTEIN
Destined for use in cell or for transport
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PROTEIN TRANSLATION

• m-RNA GOES THRU RIBOSOME.
• RIBOSOME IS r-RNA,CODE THREADS THRU RIBOSOME.
• AREA OF RIBOSOME BOUND TO tRNA

• 20 TYPES OF AA
• ANTICODON ON ONE END OF t-RNA.
• AA ON OTHER END OF t-RNA
• AA ATTACH TO EACH OTHER IN PEPTIDE BOND
• FORM PROTEINS

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The triplet code
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Role of Ribosome
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mRNA ? Ribosome

• mRNA leaves the nucleus via nuclear pores.
• Ribosome has 3 binding sites for tRNAs
• A-site position that aminoacyl-tRNA molecule
  binds to vacant site
• P-site site where the new peptide bond is
  formed.
• E-site the exit site
• Two subunits join together on a mRNA molecule
  near the 5 end.
• The ribosome will read the codons until AUG is
  reached and then the initiator tRNA binds to the
  P-site of the ribosome.
• Stop codons have tRNA that recognize a signal to
  stop translation. Release factors bind to the
  ribosome which cause the peptidyl transferase to
  catalyze the addition of water to free the
  molecule and releases the polypeptide.

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Purpose of tRNA

• The proper tRNA is chosen by having the
  corresponding anticodon for the mRNAs codon.
• The tRNA then transfers its aminoacyl group to
  the growing peptide chain.
• For example, the tRNA with the anticodon UAC
  corresponds with the codon AUG and attaches
  methionine amino acid onto the peptide chain.

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tRNAs are specific carriers of amino acids

• Aminoacyl-tRNA synthetases attach specific amino
  acids to heat-stable tRNA molecules

ATP
Amino acid
Amino acid
3
5
aminoacyl-tRNA synthetase
tRNA e.g. tRNAPhe
aminoacylated tRNA e.g. Phe-tRNAPhe
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RNA ? Protein Translation

• Ribosomes and transfer-RNAs (tRNA) run along the
  length of the newly synthesized mRNA, decoding
  one codon at a time to build a growing chain of
  amino acids (peptide)
• The tRNAs have anti-codons, which complimentarily
  match the codons of mRNA to know what protein
  gets added next
• But first, in eukaryotes, a phenomenon called
  splicing occurs
• Introns are non-protein coding regions of the
  mRNA exons are the coding regions
• Introns are removed from the mRNA during splicing
  so that a functional, valid protein can form

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Translation, continued

• Catalyzed by Ribosome
• Using two different sites, the Ribosome
  continually binds tRNA, joins the amino acids
  together and moves to the next location along the
  mRNA
• 10 codons/second, but multiple translations can
  occur simultaneously

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